Ozgun Le Roux

Despite over 50 possible oncogenic RAS mutations, cancers tend to have a bias towards a specific subset, often unique to each cancer type. As RAS mutation occur early, if not being the first mutations, these mutation patterns ostensibly reflect the selection of specific RAS mutations in each tissue to initiate tumorigenesis. Different RAS mutations can affect the level of active oncprotein and/or the type of pathways activated thereof. To attempt to determine the contribution of these two forms of signaling on the process of RAS mutation patterns in cancer, we generated four conditional murine Kras alleles containing one of two distinct oncogenic mutations, G12D or Q61R, encoded by native rare or common codons to induce low or high protein expression, respectively. These four alleles were globally activated throughout the mouse, revealing different tissue susceptibilities to the oncogenic Kras alleles. We observed that hematolymphopoietic lesions were preferentially initiated by the...

Despite over 50 possible oncogenic RAS mutations, cancers tend to have a bias towards a specific subset unique to each cancer type. As RAS mutations could arise from selection of an ideal level (quantitative) or type (qualitative) of oncogenic signaling, we generated four conditional Kras alleles with different oncogenic mutations, G12D or Q61R, coded with native rare or common codons to induce low or high protein expression to separate these two types of signaling. These alleles were crossed into a Rosa26-CreER background and globally activated, revealing tissues sensitive to quantitative signaling regulated by the RAS biochemical activity, qualitative signaling regulated by the specific mutation, and either both or none of these two types of signaling. Bulk RNAseq analysis further revealed that each allele induced a different cellular response, with the weakest allele induce transcriptional features of a plastic state while the strongest allele had all the transcriptional hallmark...

Because amino acid activation is rate-limiting for uncatalyzed protein synthesis, it is a key puzzle in understanding the origin of the genetic code. Two unrelated classes (I and II) of contemporary aminoacyl-tRNA synthetases (aaRS) now translate the code. Observing that codons for the most highly conserved, Class I catalytic peptides, when read in the reverse direction, are very nearly anticodons for Class II defining catalytic peptides, Rodin and Ohno proposed that the two superfamilies descended from opposite strands of the same ancestral gene. This unusual hypothesis languished for a decade, perhaps because it appeared to be unfalsifiable. The proposed sense/antisense alignment makes important predictions. Fragments that align in antiparallel orientations, and contain the respective active sites, should catalyze the same two reactions catalyzed by contemporary synthetases. Recent experiments confirmed that prediction. Invariant cores from both classes, called Urzymes after Ur = ...

The ability to translate three nucleotide sequences, or codons, into amino acids to form proteins is conserved across all organisms. All but two amino acids have multiple codons, and the frequency that such synonymous codons occur in genomes ranges from rare to common. Transcripts enriched in rare codons are typically associated with poor translation, but in certain settings can be robustly expressed, suggestive of codon-dependent regulation. Given this, we screened a gain-of-function library for human genes that increase the expression of a GFPrare reporter encoded by rare codons. This screen identified multiple components of the mitogen activated protein kinase (MAPK) pathway enhancing GFPrare expression. This effect was reversed with inhibitors of this pathway and confirmed to be both codon-dependent and occur with ectopic transcripts naturally coded with rare codons. Finally, this effect was associated, at least in part, with enhanced translation. We thus identify a potential re...

Despite multiple possible oncogenic mutations in the proto-oncogene KRAS, unique subsets of these mutations are detected in different cancer types. As KRAS mutations occur early, if not being initiating, these mutational biases are ostensibly a product of how normal cells respond to the encoded oncoprotein. Oncogenic mutations can impact not only the level of active oncoprotein, but also engagement with effectors and other proteins. To separate these two effects, we generated four novel inducible Kras alleles encoded by the biochemically distinct mutations G12D versus Q61R encoded by native (nat) rare versus common (com) codons to produce either low or high protein levels. Each allele induced a distinct transcriptional response in normal cells. At one end of the spectrum, the KrasnatG12D allele induced transcriptional hallmarks suggestive of an expansion of multipotent cells, while at the other end, the KrascomQ61R allele exhibited all the hallmarks of oncogenic stress and inflammat...

The ability to translate three nucleotide sequences, or codons, into amino acids to form proteins is conserved across all organisms. All but two amino acids have multiple codons, and the frequency that such synonymous codons occur in genomes ranges from rare to common. Transcripts enriched in rare codons are typically associated with poor translation, but in certain settings can be robustly expressed, suggestive of codon-dependent regulation. Given this, we screened a gain-of-function library for human genes that increase the expression of a GFPrare reporter encoded by rare codons. This screen identified multiple components of the mitogen activated protein kinase (MAPK) pathway enhancing GFPrare expression. This effect was reversed with inhibitors of this pathway and confirmed to be both codon-dependent and occur with ectopic transcripts naturally coded with rare codons. Finally, this effect was associated, at least in part, with enhanced translation. We thus identify a potential regulatory module that takes advantage of the redundancy in the genetic code to modulate protein expression.

Endotoxin (LPS)-induced changes in histone lysine methylation contribute to the gene-specific transcription for control of inflammation. Still unidentified are the chromatin regulators that drive the transition from a transcriptional-repressive to a transcriptional-active chromatin state of pro-inflammatory genes. Here, using combined approaches to analyze LPS-induced changes in both gene-specific transcription and protein secretion to the extracellular compartment, we characterize novel functions of the lysine demethylase PHF8 as a pro-inflammatory, gene-specific chromatin regulator. First, in the LPS-induced, acute-inflamed macrophages, PHF8 knockdown led to both a reduction of pro-inflammatory factors and an increase in a transcriptional-repressive code (H3K9me2) written by the methyltransferase G9a. Through unbiased quantitative secretome screening we discovered that LPS induces the secretion of a cluster of PHF8-dependent, 'tolerizable' proteins that are related to diverse extracellular pathways/processes including those for the activation of adaptive immunity. Specifically, we determined that PHF8 promotes T-cell activation and proliferation, thus providing the first link between the epigenetic regulation of inflammation and adaptive immunity. Further, we found that, in the acute-inflamed macrophages, the acute-active PHF8 opposes the H3K9me1/2-writing activity of G9a to activate specific protein secretions that are suppressed by G9a in the endotoxin-tolerant cells, revealing the inflammatory-phenotypic chromatin drivers that regulate the gene-specific chromatin plasticity. Activation of inflammation, the key host innate immune response to microbial challenge 1 , is a double-edged sword; it protects the host from infection and cellular damage, yet, its deregulation contributes directly to various inflammation-associated pathologies 2,3. Previous studies indicated that control of inflammation is achieved by endotoxin-or lipopolysaccharide (LPS)-induced gene-specific chromatin modifications. The landscape of promoter chromatin modifications is differentially programmed for a class of pro-inflammatory or " tolerizeable " (T-class) genes, in correlation with either acutely or chronically inflamed nature or " inflammatory-phenotype " of the stimulated cells 4. However, how inflammation-phenotypic plasticity is regulated within the chromatin of pro-inflammatory genes is poorly understood. The properties of histones, the core components of chromatin, can be altered by different post-translational modifications (PTMs) that specify whether the promoter of associated gene is in an open/active or closed/

Aminoacyl-tRNA synthetases (aaRS) catalyze both chemical steps that translate the universal genetic code. Rodin and Ohno offered an explanation for the existence of two aaRS classes, observing that codons for the most highly conserved Class I active-site residues are anticodons for corresponding Class II active-site residues. They proposed that the two classes arose simultaneously, by translation of opposite strands from the same gene. We have characterized wild-type 46-residue peptides containing ATP binding sites of Class I and II synthetases, and those coded by a gene designed by Rosetta to encode the corresponding peptides on opposite strands. Catalysis by WT and designed peptides is saturable, and the designed peptides are sensitive to active-site residue mutation. All have comparable apparent second-order rate constants 2.9 - 7.0E-3 M-1s-1, or ~750,000 - 1,300,000 times the uncatalyzed rate. The activities of the two complementary peptides demonstrate that the unique informati...

The emergence of polypeptide catalysts for amino acid activation, the slowest step in protein synthesis, poses a significant puzzle associated with the origin of biology. This problem is compounded as the 20 contemporary aminoacyl-tRNA synthetases belong to two quite distinct families. We describe here the use of protein design to show experimentally that a minimal class I aminoacyl-tRNA synthetase active site might have functioned in the distant past. We deleted the anticodon binding domain from tryptophanyl-tRNA synthetase and fused the discontinuous segments comprising its active site. The resulting 130 residue minimal catalytic domain activates tryptophan. This residual catalytic activity constitutes the first experimental evidence that the conserved class I signature sequences, HIGH and KMSKS, might have arisen in-frame, opposite motifs 2 and 1 from class II, as complementary sense and antisense strands of the same ancestral gene.